In 1987, an until then unknown disease in pigs was detected in North America, from where it later spread to Canada (Hill, H.; In: Proceedings of the Mystery Swine Disease Committee meeting, Oct. 6, 1990, Denver, Colo., Livestock Conservation Institute, Madison Wis, U.S.A., Keffaber et al; Am. Assoc. Swine Pract. Newsletter 1: 1-9 (1989)). The disease, which was characterized by the fact that it induced both abortion and respiratory disease, was first called Mystery Swine Disease (MSD). Nowadays, in America and Canada, the disease is also known as Swine Infertility and Respiratory Syndrome (SIRS).
Since 1990, the disease has been found in Europe, where it first caused outbreaks in Germany, followed by outbreaks in the Netherlands and Belgium, and the disease is now spreading through Europe. In Europe, the disease is commonly known as Porcine Reproductive Respiratory Syndrome (PRRS), and as Porcine Epidemic Abortion and Respiratory Syndrome (PEARS). Currently the disease is world-wide referred to as PRRS.
The pathology is not restricted to abortion and respiratory disease. Other symptoms associated with the disease are: off feed, anorexia, and bluish discolorations of the extremities, especially the ears.
The pathogenic effects of the disease with respect to both abortion and respiratory disease have extensively been described in the comprehensive review by Christianson et al. (Swine Health and Production 2: 10-28 (1994)).
The causative agent of the disease is now known to be a small enveloped RNA virus belonging to the group of Arteriviridae. Observations made by Wensvoort et al. (J. Vet. Diagn. Invest 4; 134-138 (1992) and by Murtaugh et al. (Arch. Virol. 140: 1451-1460 (1995) made it unequivocally clear that two fully different (sero)types of the virus exist: the American and the European (sero)type.
The European type of this virus has been described by Wensvoort et al. (The Vet. Quarterly; 13:121-130 (1991)). A strain of this European type, called the "Lelystad Virus" (LV) has been deposited with the Institut Pasteur, Paris, France under no. I-1102, in connection with PCT WO 92/21375 by the Central Veterinary Institute, Lelystad, The Netherlands. Another European strain has been described in EPA no. 91.202.646.5, and was deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris France under no. I-1140. This European strain has recently been described by Conzelmann et al. (Virology 193: 329-339 (1993)).
The American type of the virus has been described by Benfield et al. (J. Vet. Diagn. Invest. 4: 127-133 (1992)). A strain of the American type has been deposited with the ATCC under no VR-2332, and is mentioned in PCT WO 93/03760 and European Patent Application 0.529.584. An attenuated strain of the American serotype has been described in European Patent Application 0.529.584. This strain is directly derived from the deposited American VR-2332 strain. Therefore, animals vaccinated with this strain will not obtain an efficient protection against infection with European strains. This made it necessary to develop a live attenuated strain of the European serotype to be used as a basis for vaccines against European serotypes.
In European Patent Application No. EP 0.676.467 live attenuated strains of the European serotype are disclosed for the first time. Examples of attenuated strains of the European serotype are the virus strains PRRS C and PRRS D, as deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris, France, under the Accession number I-1387 (Strain D) and number I-1388 (Strain C).
These strains are effective as a basis for live attenuated vaccines against PRRSV. Nevertheless they still have a certain level of virulence left. Therefore, alternatively attenuated PRRSV strains and methods for the preparation of such viruses are desirable.
It is an object of the present invention to provide live attenuated PRRS-virus strains of a European serotype having the desired feature of alternative attenuation characteristics compared to the existing attenuated PRRS virus strains. It is another object of the present invention to provide methods for the preparation of such live attenuated PRRS virus strains.
These objectives are met by the present invention which provides live attenuated PRRS viruses of a European serotype that have the unique feature that they are not infectious to macrophages. It is shown in the Examples that such strains show a more attenuated character in pigs, than the parent strains that still are infectious to macrophages.
The wording "non-infectious to macrophages" has to be understood as follows: a dose of &lt;10.sup.4 Tissue Culture Infectious Doses (TCID).sub.50 according to the present invention does not give a visible CPE on a macrophage culture even at 7 days after infection. A typical macrophage-infectious PRRSV field strain would give a full CPE with infectious particle doses from 1 TCID.sub.50 and up.
Viruses of the European serotype are characterized in that they give a higher serum titre in an Immunoperoxidase Monolayer Assay with a panel of antisera against the European PRRS virus LV (CDI-NL-2.91; as deposited with the Institut Pasteur under I-1102) compared to a reaction with a panel of antisera against the American PRRS virus SIRSV (ATCC VR-2332).
It is another feature of the invention that it provides methods for the preparation of live attenuated PRRS strains according to the invention. Currently, all European PRRSV strains are obtained from macrophages of infected animals, and then if possible kept on MA 104 cells or clones thereof for propagation. The methods according to the present invention relate to the adaptation of MA 104-grown PRRS virus to non-MA 104 cells. These non-MA 104 cells may, for example be cells with a limited life span, or established immortalized cell lines. In general, the non-MA 104 cells will be mammalian cells. Mammalian non-MA 104 cells are, for example, general purpose cell lines such as BHK cell lines, CRFK cell lines and Madin Darby Bovine Kidney cell lines or, for example porcine cells such as Swine testicle cells or Swine kidney cells, or they may be non-MA 104 primate cells such as VERO cells. Adapting PRRSV to cells other than the natural host cell comprises standard methods that can be performed as follows: a specific PRRS virus isolate is grown on a culture containing porcine cells or other susceptible cells of non-porcine origin, at which the virus can be multiplied, such as MA 104 cells. After growth, the PRRSV is harvested by collecting the cell culture fluids and/or cells followed by passage of the cell culture fluids and/or cells onto another cell type for adaptation. Strains that have first been adapted to one non-MA 104 cell line can additionally be adapted to another non-MA 104 cell line for further attenuation. If desired, this adaptation process can be repeated on still other non-MA 104 cells. Adaptation to growth at different temperatures may also be part of the attenuation process. After passage to non-MA 104 cells, the non-macrophage-infectious character can be obtained after a small number of passages. A number of 5 passages, usually the number of passages necessary to obtain a master seed virus, often suffices to obtain non-macrophage-infectious viruses according to the invention.
In a preferred form, the method uses non-MA 104 primate cells as the cells for the adaptation. It is clear that after adaptation to non-MA 104 primate cells any further adaptation can be done on any other suitable cells.
In a more preferred form, the method uses Vero cells as the non-MA 104 primate cells for the adaptation.
Checking for the non-macrophage-infectious character of the so obtained live attenuated strains can easily be done by testing the absence of growth of the so obtained strains in vitro on macrophages. Standard techniques for both the preparation of macrophage cultures and propagation of macrophage-infectious virus are known in the art, and have, for example, been described in WO 93/07898. A simple test system based on macrophages isolated and grown under standard conditions will do as a test system to discriminate live attenuated PRRS strains according to the invention from other live attenuated PRRS viruses that still retain their macrophage-infectious character. One possible test system is described in Example 2.
The attenuated character of PRRSV strains according to the present invention can be illustrated departing from two strains: I-1140, a pathogenic PRRSV strain isolated in 1991 in the Netherlands, and I-1387, a low pathogenic PRRSV field strain (both strains deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris France under their respective number). These parent strains are infectious for macrophages. Of these parent strains, attenuated non-macrophage-infectious strains according to the invention were made as described in Example 1. The macrophage-infectious character was determined as described in Example 2.
The following strains were available for tests:
1. I-1140 88.sup.th passage on MA 104 cells PA1 2. I-1140 61.sup.th passage on Vero cells PA1 3. I-1387 20.sup.th passage on MA 104 cells PA1 4. I-1387 49.sup.th passage on Vero cells
The following shows the test results checking for the infection-characteristics of these strains:
TABLE 1 ______________________________________ Comparison of strains according to the invention and high passage numbers of the parent strains Cell-line Strain Macrophage Vero MA104 ______________________________________ 1 +++ --- +++ 2 --- +++ +++ 3 +++ --- +++ 4 --- +++ +++ ______________________________________
Also very suitable as parent strains for attenuation according to the present invention are PRRS viruses that are already attenuated in another (i.e. not relating to macrophage infectivity) aspect. Such viruses have been described in European Patent Application No. EP 0.676.467, mentioned above. These virus strains belong to a European serotype and they are reactive with monoclonal antibody A27 as produced by hybridoma A27 deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris France under No. I 1401, but not with monoclonal antibody A35 as produced by hybridoma A35 deposited with the Institut Pasteur at Paris France under No. I 1402. If such viruses are made non-infectious to macrophages according to methods of the present invention, they are even more safe since then their attenuated character relies on two or more different aspects, one of which is the non-macrophage-infectious character. Therefore, in a more preferred embodiment of the present invention, the live attenuated PRRSV according to the invention has the additional characteristics of being reactive with monoclonal antibody A27 as produced by hybridoma A27 deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris France under No. I 1401, but not with monoclonal antibody A35 as produced by hybridoma A35 deposited with the Institut Pasteur at Paris France under No. I 1402.
Since the non-macrophage-infective strain according to the present invention derived from the I-1387 strain is so highly attenuated after passage over Vero cells, this live attenuated strain is extremely safe when administered to animals (see Example 2 and further). Therefore, in another more preferred embodiment of the present invention, the live attenuated PRRSV is of the strain that is deposited by AKZO Nobel NV at the Collection Nationale de Cultures de Microorganismes of the Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15 under Accession Number I-1758. This strain was given the Identification Reference "PRRSV strain DV" and thus will also be referred to as strain DV in this Specification.
The live attenuated viruses according to the present invention are, as a result of their inability to infect macrophages, a very suitable basis for vaccines for the following reason: These strains are not infectious to the macrophage. The macrophage is the primary target cell of wild-type PRRSV and one of the key cells of the immune system. Nevertheless, they still have retained their ability to trigger the immune system. Therefore, contrary to vaccines based on known live attenuated strains of the European serotype as described in European Patent Application No. EP 0.676.467, vaccines comprising the live attenuated viruses according to the present invention trigger the immune system without at the same time impairing it.
Thus, vaccines based on the strains of the present invention provide safety of a different kind than vaccines based on the known attenuated strains.
Therefore, in still another embodiment, the present invention provides vaccines for the protection of pigs against PRRSV infection, that are based on the live non-macrophage-infectious attenuated PRRS strains described above.
Due to the safety of the non-macrophage-infectious viruses, vaccines based on these viruses may typically contain 10.sup.3 -10.sup.10 live virus particles.
Vaccines according to the present invention may comprise a pharmaceutically acceptable carrier. One possible carrier is a physiological salt solution. Another pharmaceutically acceptable carrier is for instance the tissue culture fluid used for sustaining the cell growth, in which the viruses are released from the infected cells.
An adjuvant and if desired one or more emulsifiers such as Tween and Span may also be incorporated in the live attenuated vaccine according to the invention. Suitable adjuvants are for example vitamin E acetate solubilisate, aluminium hydroxide, -phosphate or -oxide, (mineral) oil emulsions such as Bayol and Marcol52, and saponins. Incorporation of the antigens in Iscoms is also a possible way of adjuvation.
The vaccine according to the invention is produced preferably in a freeze-dried form. It is advantageous to add a stabilizer to live attenuated viruses, particularly if a dry composition of live viruses is prepared by lyophilisation. Suitable stabilizers are, for example, SPGA (Bovamik et al., J. Bacteriology 59, 509, 1950), carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein), or degradation products thereof, and buffers (such as alkali metal phosphates). If desired, one or more compounds with adjuvant activity as described above can also be added.
A vaccine according to the invention may be administered by intramuscular or subcutaneous injection or via intranasal, intratracheal, oral, cutane, percutane/intradermal or intracutane administration. A very convenient way of administration is intradermal or intramuscular administration. Therefore, in a preferred embodiment, the vaccine according to the invention comprises a carrier that is suitable for intradermal or intramuscular application. A physiological salt solution is, for example a simple and suitable carrier for intradermal or intramuscular application.
The vaccine according to the invention can be administered to pigs depending on the vaccination history of the sows at 1, 3, 6 or 10 weeks of age, to sows before mating and/or up to 6 weeks before farrowing (booster vaccination), or to boars each half a year (boosters).
In a preferred form, the vaccine of the present invention comprises, next to live attenuated PRRS virus, another unrelated (i.e. non-PRRSV) attenuated or inactivated pathogen or antigenic material from another pathogen. Such a pathogen may be a bacterium or a parasite, but may also be of viral origin. Usually, the unrelated pathogen or antigenic material thereof will be a porcine pathogen. A vaccine according to the invention that also comprises such an additional attenuated or inactivated pathogen or antigenic material from another pathogen has the advantage that it induces protection against several infections at the same time. Antigenic material is understood to be material that is capable of inducing an immunogenic response. Examples of antigenic material are proteins, polysaccharides and lipopolysaccharides.
In a more preferred form, the pathogen is selected from the group of Pseudorabies virus, Porcine influenza virus, Porcine parvovirus, Transmissible gastroenteritis, Rotavirus, Escherchia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.
A vaccine according to the invention may be derived from any live attenuated PRRS virus isolate of a European serotype according to the present invention. The deposited PRRSV strain DV, developed by using methods according to the present invention has all the characteristics that make it a very suitable vaccine strain. Thus in a preferred form, the vaccine is derived from PRRSV strain DV, deposited at the Institut Pasteur under Accession Number I-1758.
In again still another embodiment, the present invention provides methods for the preparation of a live attenuated vaccine for combating PRRS. A convenient way of obtaining a vaccine based on live attenuated PRRSV according to the invention comprises admixing a suitable amount of virus according to the invention with a pharmaceutically acceptable carrier.